Contemporary Enzyme-Based Methods for Recombinant Proteins In Vitro Phosphorylation

Phosphorylation is a reversible, enzyme-controlled posttranslational process affecting approximately one-third of all proteins in eukaryotic cells at any given time. Any deviation in the degree and/or site of phosphorylation leads to an abnormal conformation of proteins, resulting in a decline or loss of their function. Knowledge of phosphorylation-related pathways is essential for understanding the understanding of the disease pathogenesis and for the design of new therapeutic strategies. Recent availability of various kinases at an affordable price differs in activity, specificity, and stability and provides the opportunity of studying and modulating this reaction in vitro. We can exploit this knowledge for other applications. There is an enormous potential to produce fully decorated and active recombinant proteins, either for biomedical or cosmetic applications. Closely related is the possibility to exploit current achievements and develop new safe and efficacious vaccines, drugs, and immunomodulators. In this review, we outlined the current enzyme-based possibilities for in vitro phosphorylation of peptides and recombinant proteins and the added value that immobilized kinases provide

Controlled proteolysis of normal and pathological prion protein in a microfluidic chip.

A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactor's performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated

Systèmes microfluidiques de particules magnétiques auto-assemblées; Application à la séparation d’ADN et à la digestion de protéines.

Nous présentons deux systèmes microfluidiques d’analyses biologiques fondés sur l’auto-organisation de billes magnétiques sous un champ magnétique. Dans le premier dispositif, les billes sont organisées en colonnes dans une structure de réseau quasi-hexagonal utilisé comme matrice pour réaliser et étudier la séparation de longs fragments d’ADN. Dans le second on a greffé de la trypsine sur les billes magnétiques, que l’on immobilise dans un canal microfluidique entre deux aimants. Différentes protéines sont alors digérées à travers cet amas de billes et les produits de digestion analysés par spectroscopie de masse ou par électrophorèse capillaire